Exhaled endogenous particles contain lung proteins.
Biological changes caused by airway inflammation are difficult to monitor. Currently, the most efficient methods for sampling RTLF are invasive, such as bronchoalveolar lavage (BAL), or semi-invasive, such as induced sputum. These methods are not applicable for studies of large populations, or for repeated sampling and screening methods. Therefore, the noninvasive sampling of exhaled breath condensate (EBC) has become a rapidly growing field in respiratory analysis; however, EBC has a number of serious methodologic difficulties, e.g., correction for dilution and lack of standardization, such that data produced by different groups can vary considerably (7-9).
We have recently developed a novel, noninvasive technique for collecting nonvolatile material from the respiratory system (10). The individuals exhale into the sampling device, which uses a 3-stage impactor to collect endogenous particles in exhaled air (PEx) for subsequent chemical analysis. How PEx are formed is not yet fully understood, but one likely mechanism is rupture of the RTLF film during airway reopening after airway closure (11). In previous work, we have detected phospholipid species, such as phosphatidylcholine and phosphatidylglycerol, that were consistent with an RTLF composition (10). The observed phospholipid composition of PEx was consistent with that of lung surfactant, further supporting RTLF as the origin of PEx.
To evaluate the potential of PEx for respiratory research requires establishing the molecular composition of the exhaled endogenous particles. We present the first proteomic study of PEx. By analyzing the protein composition of PEx, we aim to verify their origin as being the RTLF and to evaluate them as a source for biomarkers of respiratory diseases.
Materials and Methods
CHEMICALS AND REAGENTS
DL-Dithiothreitol [greater than or equal to] 99%>, iodoacetamide Sigma Ultra, and trifluoroacetic acid reagent grade [greater than or equal to] 98%> were purchased from Sigma-Aldrich (http://www.sigmaaldrich. com). Acetonitrile HPLC gradient grade was purchased from Fisher Scientific (http://www.fishersci.com). Sequencing-grade trypsin was purchased from Promega (http://www.promega.com). Ammonium bicarbonate Puriss (N[H.sub.4]HC[O.sub.3]) was purchased from Riedel-de Haen (http://www.riedeldehaen.com). PBS (0.14 mol/L NaCl; 0.0027 mol/L KCl; and 0.01 mol/L phosphate buffer; pH 7.4) was purchased from Medicago (http://www.medicago.se). All chemicals and precast gels for the 1-dimensional gel electrophoresis were purchased from Invitrogen (http://www.invitrogen. com). High-purity water was used for all experiments (Milli-Q Plus; Millipore, http://www.millipore.com).
The study included 12 healthy volunteers. These individuals had a mean age of 36 years (range, 28-57 years), and 8 (67%) of the volunteers were female. The study was approved by the local ethics committee of the Sahlgrenska Academy of Gothenburg, and all participants gave their informed consent.
PEx were collected onto a silicon plate with a custom-built sampling device previously described by Almstrand et al. (10). Forced exhalations were performed into the sampling device, where the particles were simultaneously counted by an optical counter and collected inside a 3-stage impactor. Between forced exhalations, the participants inhaled particle-free air tidally through a particle filter.
There were 2 sampling sessions. Pooled samples from 6 healthy individuals were collected in the first session, for a 3000-L total volume of exhaled air. In the second session, pooled samples from 10 healthy individuals were collected, for a total volume of 4400 L.
We collected pooled samples by having the participants take turns exhaling for 25 min (100-200 L of air) onto the same sampling plate. Four of the participants from the first session were included in the second session. All participants rinsed their mouths with purified water and breathed particle-free air for 2 minutes before sampling. Each participant wore a nose clip throughout the sampling procedure. The silicon plates containing the exhaled particles were stored at -20 [degrees]C before analysis. For PEx analysis, the proteins were extracted from the silicon plates with NuPAGE sample buffer (Invitrogen) and PBS.
A control sample of room air was collected by drawing room air into the sampling device for 7.5 h. Particles collected in this sample were analyzed with the same procedure used for the pooled samples.
TOTAL PROTEIN CONTENT
The total protein content of PEx was measured with a CBQCA Protein Quantification Kit (Molecular Probes Europe) and a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon).
Proteins in PEx extracts were separated by SDS-PAGE before tryptic digestion and LC-MS analysis. Proteins were separated under reducing conditions on precast 1-dimensional gels (NuPAGE 4%-12% Bis-Tris Gel) using NuPAGE MES SDS Running Buffer and the XCell SureLock[TM] Mini-Cell system. SeeBlue[R] Plus2 Pre-Stained Standard protein mix was used as molecular weight standards, and proteins were stained with the Colloidal Coomassie Blue Staining Kit.
IN-GEL DIGESTION AND PEPTIDE EXTRACTION
The gel from the first sample was cut into 17 slices, including 1 control slice from a blank region of the gel. The slices were cut into 1 mm cubes and destained in acetonitrile/100 mmol/L N[H.sub.4]HC[O.sub.3] (45:55 volume ratio) for1honashaker at room temperature. The solution was removed, the gel samples were dehydrated by incubation with 75 [micro]L of ethanol for 5 min, and the ethanol was removed. Proteins were reduced and alkylated by incubation with 10 mmol/L dithiothreitol for 30 min at 37 [degrees]C, followed by incubation with 55 mmol/L iodoacetamide for 30 min in the dark at room temperature. After each step, the supernatants were removed, and the samples were dehydrated by incubating them with 75 [micro]L ethanol for 5 min and then removing the ethanol. For trypsinolysis, 15 [micro]L trypsin solution (10 ng/[micro]L trypsin in 50 mmol/L N[H.sub.4]HC[O.sub.3]) was added to the dry gel pieces. After a 30-min incubation at 4 [degrees]C, additional volumes of 50 mmol/L N[H.sub.4]HC[O.sub.3] sufficient to cover the gel pieces were added to the samples, followed by incubation overnight at 37 [degrees]C. Peptides were extracted with 5 mL/L trifluoroacetic acid in 2 mmol/L n-octyl-[beta]-D-glucopyranoside for 30 min. The eluates were lyophilized in a SpeedVac (Savant) and stored at -20 [degrees]C until LC-MS analysis.
The gel from the second sample was cut into 15 slices. The individual slices were reduced, alkylated, and digested with trypsin as described above with the aid of a Biomek 2000 Laboratory Automation Workstation (Beckman Coulter).
Peptide mixtures were analyzed by reversed-phase nanoscale capillary liquid chromatography coupled to electrospray ionization mass spectrometry. The samples were injected onto a 15-cm reversed-phase column (3-[micro]m ReproSil-Pur C18 AQ media; Dr. Maisch GmbH) packed in an uncoated, fused silica emitter (PicoFrit: 15-cm length, 75-[micro]m inner diameter, 8-[micro]m tip inner diameter; New Objective) according to the procedure of Ishihama et al. (12). Liquid chromatography was performed on a CapLC System (Waters) fitted with a passive flow-splitter to reduce the flow to 150 -250 nL/min. The liquid chromatography system was connected to a linear quadrupole ion trap-Fourier transform ion cyclotron resonance (LTQ-FT ICR) mass spectrometer (Thermo Scientific) operated in the data-dependent mode. Survey spectra in the m/z range of 350-2000 were acquired in the ICR cell, and the 5 detected ions with the highest intensities were selected in order of decreasing intensity for fragment ion mass spectrometry ([MS.sup.2]) analysis in the LTQ. Singly charged ions were excluded from the [MS.sup.2] analysis. Ions within a window of 5 ppm around the m/z values of the ions selected for [MS.sup.2] analysis were dynamically excluded for a time period of 45 s. The data from the first sample were converted to centroid data with the instrument manufacturer's software. The data from the second sample were stored as profile data, and monoisotopic m/z values were extracted with Mascot Distiller software (MatrixScience).
Proteins were identified with the Mascot software by searching the IPI human database (Mascot version 2.3 and IPI version 3.72, 86 392 sequences). The following settings were used for the searches: mass error tolerance for the precursor ions, 10 ppm for centroid data and 5 ppm for raw data; mass error tolerance for the fragment ions, 0.4 Da; fixed modification, carbamidomethylation; variable modification, methionine oxidation; number of missed cleavage sites, 1; type of instrument, FT-ICR.
A protein was considered identified if it had a minimum of 2 peptides with a Mowse score [greater than or equal to] 20. For proteins identified from <2 peptides with Mowse scores >20, peptide fragment spectra were evaluated manually to accept or reject the identification.
TOTAL PROTEIN CONTENT
Particles were collected from 300 L of exhaled air. The amount of total protein in the sample was 100 ng, i.e., 0.33 ng of PEx per liter of exhaled air. The total mass of particles calculated for this sample was 440 ng, which corresponds to a protein content of 23% in PEx. This result is in agreement with the 10% protein content of surfactant previously reported (3). The result from the analysis of total protein content was used to estimate the total protein content in the 2 samples used for protein characterization, according to the volumes of air sampled.
We identified 124 proteins in the 2 PEx samples, 103 (83%) of which have previously been identified in BAL (5, 6). The first sample was collected from 3000 L of exhaled air containing approximately 1 [micro]g total protein; the second sample obtained from 4400 L of exhaled air contained approximately 1.5 [micro]g total protein. We identified 32 proteins in the first sample and 116 in the second sample, with 24 of the proteins being shared by the 2 samples. Among the shared proteins were some of the most prominent blood and BAL proteins: albumin, serotransferrin, surfactant protein A (SP-A), [[alpha].sub.1]-antitrypsin, and immunoglobulins (13). Table 1 lists the identified proteins.
The proteins were sorted according to their cellular component annotations in ProteinCenter[TM], a Web-based data-interpretation tool (Proxeon). Many of the identified proteins were associated with multiple cellular locations (Table 2), with extracellular proteins constituting the largest group. Ninety-eight (79%) of the 124 identified proteins were annotated as extracellular. Cytoplasm and membrane were other abundantly represented cellular locations, accounting for 63% and 48%, respectively, of the identified proteins. Many of the cytoplasmic and membrane proteins were additionally-annotated as extracellular proteins, specifically 74% of the cytoplasmic proteins and 75% of the membrane proteins.
The high number of extracellular proteins in PEx, which constituted almost 80% of the identified proteins, is in strong agreement with our assumption that PEx should contain mainly secreted and extracellular proteins, similar to those of RTLF.
RELATIVE QUANTIFICATION OF THE IDENTIFIED PROTEINS
We used the exponentially modified protein abundance index (emPAI) to obtain an approximate, relative quantification of the identified proteins. The emPAI is a label-free protocol that estimates the relative quantities of the proteins in a mixture from the protein coverage revealed by the peptide matches in a database search (14). The contributions of the 9 most abundant proteins were: albumin, 26%; surfactant proteins, 13% (including SP-A, 12%); various immunoglobulins, 14%; serotransferrin, 4%; Clara cell protein (CC16), 4%; lysozyme C, 2%; proteases and inhibitors, 4% (including [[alpha].sub.1]-antitrypsin, 2%); annexins, 1%; and complement factors, 1%.
PROTEINS IDENTIFIED IN ROOM AIR
Six of the 40 proteins identified in room air were also identified in at least one of the samples, indicating possible contamination of the sample. The identified possible contaminants were: filaggrin 2, glyceraldehyde-3-phosphate dehydrogenase, deleted in malignant brain tumors 1 protein, isoform DPI of desmoplakin, prolactin-inducible protein, and serpin B12. None of the major proteins mentioned above in the PEx samples were identified in room air, however.
For PEx to originate from RTLF, one would expect PEx to show similarities with BAL fluid, which is commonly used for the collection of RTLF. We used data from 2 independent BAL proteomic studies for comparison with the proteins identified in PEx (5, 6). Of the 124 identified proteins, 103 had previously been reported to occur in BAL fluid, a finding that is in good agreement with our assumption that the composition of PEx reflects that of RTLF. The majority of the identified proteins were extracellular proteins. We compared the distribution of the cellular localizations of the proteins identified in PEx with the distributions obtained in other human proteome projects: HUPO Brain, HUPO Plasma, HUPO Urine. PEx showed considerable overrepresentation of extracellular proteins compared with these projects, with 70% of the PEx proteins being extracellular, in contrast to the 20%, 16%, and 55% values obtained in the brain tissue, plasma, and urine projects, respectively. The prominent representation of extracellular proteins is in agreement with our suggestion that PEx mainly contain secreted and extracellular RTLF proteins.
The main component of lower-airway RTLF is lung surfactant, which contains surfactant proteins, SP-A being the most abundant. Our evaluation of the relative distribution of proteins in PEx with the emPAI protocol showed that the most abundant protein was albumin (26% of the total protein content). Owing to the permeability of the endothelial and epithelial barrier of the lungs, we expected a high abundance of major blood proteins. The second most abundant protein was locally produced SP-A, which constituted 12% of the total protein. The high abundance of SP-A indicates that lung surfactant is a major component of PEx, as would be expected for material collected from the lower airways. Because the protein distribution we have presented is based on an approximate quantification, confirmation with an alternative quantification method is required.
The identification of proteins typical of alveolar type II cells and Clara cells (such as SP-A, SP-B, SP-C, and CC16) further supports the hypothesis that PEx is formed in the distal airways. A number of the proteins identified in PEx, such as complement factor B, C1q, C2, C3, and C4, have previously been reported to playa role in allergic airway inflammation. Other identified proteins, such as annexins A1, A2, A3, and A5, have proved to be inhibitors of phospholipase A2. Phospholipase A2 is an enzyme responsible for the release of arachidonic acid, a precursor of inflammatory mediators (eicosanoids). Various proteases and protease inhibitors were among the identified proteins. The identification of proteins related to airway inflammation suggests that PEx measurement will enable monitoring of ongoing inflammatory processes and be of great importance in respiratory research. The complete list of identified proteins presented in Table 1 provides references to published data.
At present, EBC is the only noninvasive commercially available method for sampling material from the airways. A variety of biomarkers, including proteins, have been detected in EBC (15, 16); however, EBC protein studies have been restricted mainly to specific protein assays, e.g., cytokine immunodetection; therefore, we did not include a comparison of EBC and PEx protein compositions in this study.
With the PEx method, the material to be analyzed is sampled from exhaled air similarly to EBC sampling; however, the PEx and EBC sampling methods have substantial methodologic differences (10). EBC collection involves the condensation of exhaled matter (8), and condensation efficiencies are likely to vary, depending on the chemical composition of the exhaled particles. In PEx, the collection of exhaled particles is based on particle size and should be independent of the particle content. Particles or droplets consisting of RTLF are also collected with EBC (16); however, they are more efficiently collected with the PEx method and show no dilution. Dilution is an uncertainty factor for EBC, and is perhaps even more uncertain for BAL, in which adding external fluid to the airways makes it difficult to calculate exact concentrations (e.g., of inflammatory mediators) in the sample (8, 17). The PEx sample is dry when collected. The sample is then extracted into a well-defined volume of suitable buffer for subsequent analysis. During the collection of PEx, the particles are counted and measured according to size, making it possible to calculate the mass of the sample. This feature is important for calculating the content of the various substances in the sample. We estimated that PEx consists of approximately 23% total protein. This finding is in agreement with previous published reports, which have estimated a protein content of 10% in surfactant, which is a major component of RTLF (3).
The ability to collect and count particles simultaneously is important, not only for measuring the total amount of sampled material from the numbers and sizes of the collected particles, but also for studies of the formation of exhaled particles. Particle formation is an important avenue of investigation and should be controlled, especially in comparative studies. In the current study, we used forced exhalations because of the original observation that they increase the formation of particles compared with tidal breathing. Our recent data show, however, that airway reopening after airway closure is the main mechanism of particle formation (11). Airway-closure maneuvers will be used instead of forced exhalations in follow-up comparative studies to improve our control of particle formation among individuals and to increase the amounts of collected material.
Given that PEx are exhaled through the oral cavity, the risk of saliva contamination has to be addressed. Amylase accounts for 60% of the total protein in saliva (18) and therefore is a good marker for the degree of salivary protein contamination. Because amylase is by far the most abundant protein in saliva, detection of salivary proteins thus is possible only when amylase is observed. The absence of amylase among the identified PEx proteins is a strong indication that we had no saliva protein contamination and that all of the proteins identified in our samples originated from the airways. This important feature of the PEx method makes it possible to perform comparative proteomic studies on material sampled exclusively from the airways.
The present work is the first proteomic study of PEx aimed at providing sufficient data for evaluating PEx as a potential source of RTLF proteins. The amounts of analyzed material were rather small, 1-1.5 [micro]g, compared with those of published BAL proteomic studies (5, 6). For this reason, the proteins identified in the current study likely represent the most abundant PEx proteins, and many less abundant proteins are missing. The number of proteins identified showed a large increase, however, when we increased the volume of sampled air. An increase of 50% (3000-4400 L) from sample 1 to sample 2 generated a 260% increase in the number of identified proteins (32 to 116). Improving the PEx sampling efficiency to increase the total amount of collected material should therefore lead to the identification of additional proteins. Our comparison of the 2 protein-identification sets showed that 75% of proteins identified in the first sample were also detected in the second. Given that the proteome is a dynamic system and that the applied mass spectrometry-based protein identification strategy discriminates the detection of low-abundance proteins, the 75% overlap indicates that the major component of the PEx proteome is rather stable in healthy individuals.
A key aspect of mass spectrometry in proteome research that sets it apart from all other analytical techniques is the ability to identify large numbers of proteins in small quantities of biological samples. Limitations of the technique for candidate protein biomarker discovery include the following: (a) There is a strong bias toward the detection of abundant sample components that, considering that the concentrations of proteins in human tissues and body fluids span many orders of magnitude, precludes the detection of many low-abundance proteins. Therefore, improving the identification coverage often requires implementing extensive protein- and peptide-fractionation steps before the mass spectrometry analysis. (b) Relative quantification of individual proteins in different biological samples is time-consuming, and differential analysis is typically limited to relatively small sample sets, features that can lead to high false-discovery rates. Mass spectrometry is therefore most often used in the discovery phase. Any candidate biomarkers discovered then have to be independently confirmed via alternative targeted and quantitative methods (19).
Our data show that the PEx-collection technique should be suitable for comparative mass spectrometry-based proteomic studies that use pooled material from well-defined groups of patients for identifying candidate biomarkers associated with disease-induced alterations of RTLF protein composition.
SUMMARY AND FUTURE PERSPECTIVE
Sampling of the distal airways is important for diagnosis, monitoring, and treatment of distal airway inflammation. The development of alternative noninvasive sampling methods would greatly facilitate our understanding of the biological processes occurring in the distal airways. As a step toward this goal, PEx provide the means for the noninvasive collection of proteins from the respiratory system. Our data strongly support the conclusion that PEx reflect the composition of undiluted RTLF; these results therefore open new possibilities for comparative proteomic studies of disease-or drug-induced alterations in the protein composition of RTLF. Such comparative studies may lead to the discovery of novel inflammatory mediators that have potential as therapeutic targets or biomarkers. Similar studies carried out with BAL collected from healthy and asthmatic individuals have identified 1895 proteins, 10% of which showed different concentrations (6). Validation of the identified candidate biomarkers by alternative targeted approaches (e.g., immunoassays) could lead to the establishment of additional biomarkers applicable for monitoring of individual disease- or drug-induced alterations. Our laboratory has recently detected SP-A in PEx by ELISA (20). The advantage of PEx for monitoring alterations in the distal airways is that PEx, similarly to BAL, are collected from the target organ but that, in contrast to BAL, PEx collection is noninvasive, thus offering possibilities for studies of large populations.
Acknowledgments: We gratefully acknowledge Ulla Ruetschi, Clinical Neurochemistry Laboratory, University of Gothenburg, for discussion and assistance with experimental design, and we thank Aron Hakonen, Analytical Chemistry, University of Gothenburg, for spectrofluorometric analysis. We also thank personnel from Occupational and Environmental Medicine, University of Gothenburg, for their participation in PEx sampling.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: A.-C. Olin (principal investigator), Swedish Heart-Lung Foundation (254212008) and FORMAS (254212006), administered by the University of Gothenburg.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
(1.) Fiegel J, Clarke R, Edwards DA. Airborne infectious disease and the suppression of pulmonary bioaerosols. Drug Discov Today 2006;11:51-7.
(2.) Jarjour NN, Enhorning G. Antigen-induced airway inflammation in atopic subjects generates dysfunction of pulmonary surfactant. Am J Respir Crit Care Med 1999;160:336-41.
(3.) Goerke J. Lung surfactant. Biochim Biophys Acta 1974;344:241-61.
(4.) Nishioka T, Uchida K, Meno K, Ishii T, Aoki T, Imada Y, et al. Alpha-1-antitrypsin and complement component C7 are involved in asthma exacerbation. Proteomics Clin Appl 2008;2:46-54.
(5.) Plymoth A, Yang Z, Lofdahl CG, Ekberg-Jansson A, Dahlback M, Fehniger TE, et al. Rapid proteome analysis of bronchoalveolar lavage samples of lifelong smokers and never-smokers by micro-scale liquid chromatography and mass spectrometry. Clin Chem 2006;52:671-9.
(6.) Wu J, Kobayashi M, Sousa EA, Liu W, Cai J, Goldman SJ, et al. Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge. Mol Cell Proteomics 2005;4:1251-64.
(7.) Jackson AS, Sandrini A, Campbell C, Chow S, Thomas PS, Yates DH. Comparison of biomarkers in exhaled breath condensate and bronchoalveolar lavage. Am J Respir Crit Care Med 2007;175: 222-7.
(8.) Horvath I, Hunt J, Barnes PJ, Alving K, Antczak A, Baraldi E, et al. Exhaled breath condensate: methodological recommendations and unresolved questions. Eur Respir J 2005;26:523-48.
(9.) Rosias PP, Dompeling E, Hendriks HJ, Heijnens JW, Donckerwolcke RA, Jobsis Q. Exhaled breath condensate in children: pearls and pitfalls. Pediatr Allergy Immunol 2004;15:4-19.
(10.) Almstrand AC, Ljungstrom E, Lausmaa J, Bake B, Sjovall P, Olin AC. Airway monitoring by collection and mass spectrometric analysis of exhaled particles. Anal Chem 2009;81:662-8.
(11.) Almstrand AC, Bake B, Ljungstrom E, Larsson P, Bredberg A, Mirgorodskaya E, et al. Effect of airway opening on production of exhaled particles. J Appl Physiol 2010;108:584-8.
(12.) Ishihama Y, Rappsilber J, Andersen JS, Mann M. Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 2002;979:233-9.
(13.) Miller I, Eberini I, Gianazza E. Proteomics of lung physiopathology. Proteomics 2008;8:5053-73.
(14.) Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, Rappsilber J, et al. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 2005;4:1265-72.
(15.) Conrad D, Goyette J, Thomas P. Proteomics as a method for early detection of cancer: a review of proteomics, exhaled breath condensate, and lung cancer screening. J Gen Intern Med 2008;23:78-84.
(16.) Hunt J. Exhaled breath condensate: an overview. Immunol Allergy Clin North Am 2007;27:587-96.
(17.) Effros RM, Peterson B, Casaburi R, Su J, Dunning M, Torday J, et al. Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects. J Appl Physiol 2005;99:1286-92.
(18.) Vitorino R, Lobo MJC, Ferrer-Correira AJ, Dubin JR, Tomer KB, Domingues PM, et al. Identification of human whole saliva protein components using proteomics. Proteomics 2004;4:1109-15.
(19.) Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 2006;24: 971-83.
(20.) Larsson P, Mirgorodskaya E, Samuelsson L, Bake B, Almstrand A-C, Bredberg A, et al. Surfactant protein A and albumin in particles in exhaled air. Respir Med 2012;106:197-204.
(21.) Craig-Barnes HA, Doumouras BS, Palaniyar N. Surfactant protein D interacts with [[alpha].sub.2]-macroglobulin and increases its innate immune potential. J Biol Chem 2010;285:13461-70.
(22.) Wang P, Chintagari NR, Narayanaperumal J, Ayalew S, Hartson S, Liu L. Proteomic analysis of lamellar bodies isolated from rat lungs. BMC Cell Biol 2008;9:34.
(23.) Singh TK, Abonyo B, Narasaraju TA, Liu L. Reorganization of cytoskeleton during surfactant secretion in lung type II cells: a role of annexin II. Cell Signal 2004;16:63-70.
(24.) Kim TH, Lee YH, Kim KH, Lee SH, Cha JY, Shin EK, et al. Role of lung apolipoprotein A-I in idiopathic pulmonary fibrosis: antiinflammatory and antifibrotic effect on experimental lung injury and fibrosis. Am J Respir Crit Care Med 2010;182: 633-42.
(25.) Yao X, Fredriksson K, Yu ZX, Xu X, Raghavacharl N, Keeran KJ, et al. Apolipoprotein E negatively regulates house dust mite-induced asthma via a low-density lipoprotein receptor-mediated pathway. Am J Respir Crit Care Med 2010;182:1228-38.
(26.) Carnevali S, Luppi F, D'Arca D, Caporali A, Ruggieri MP, Vettori MV, et al. Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke. Am J Respir Crit Care Med 2006; 174:393-9.
(27.) Vasavda N, Eichholtz T, Takahashi A, Affleck K, Matthews JG, Barnes PJ, et al. Expression of nonmuscle cofilin-1 and steroid responsiveness in severe asthma. J Allergy Clin Immunol 2006;118: 1090-6.
(28.) Bolger MS, Ross DS, Jiang H, Frank MM, Ghio AJ, Schwartz DA, et al. Complement levels and activity in the normal and LPS-injured lung. Am J Physiol Lung Cell Mol Physiol 2007;292:L748-59.
(29.) Shum BO, Mackay CR, Gorgun CZ, Frost MJ, Kumar RK, Hotamisligil GS, et al. The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation. J Clin Invest 2006; 116:2183-92.
(30.) Tokunaga K, Nakamura Y, Sakata K, Fujimori K, Ohkubo M, Sawada K, et al. Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers. Cancer Res 1987; 47:5616-9.
(31.) Rottoli P, Magi B, Cianti R, Bargagli E, Vagaggini C, Nikiforakis N, et al. Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis. Proteomics 2005;5:2612-8.
(32.) Beck-Schimmer B, Madjdpour C, Kneller S, Ziegler U, Pasch T, Wuthrich RP, et al. Role of alveolar epithelial ICAM-1 in lipopolysaccharide-induced lung inflammation. Eur Respir J 2002;19:1142-50.
(33.) Adair JE, Stober V, Sobhany M, Zhuo L, Roberts JD, Negishi M, et al. Inter-alpha-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding. J Biol Chem 2009; 284:16922-30.
(34.) Poller W, Faber JP, Weidinger S, Tief K, Scholz S, Fischer M, et al. A leucine-to-proline substitution causes a defective a1-antichymotrypsin allele associated with familial obstructive lung disease. Genomics 1993;17:740-3.
(35.) Maniatis NA, Harokopos V, Thanassopoulou A, Oikonomou N, Mersinias V, Witke W, et al. A critical role for gelsolin in ventilator-induced lung injury. Am J Respir Cell Mol Biol 2009;41:426-32.
(36.) Chishimba L, Thickett DR, Stockley RA, Wood AM. The vitamin D axis in the lung: a key role for vitamin D-binding protein. Thorax 2010;65:456-62.
(37.) Rogan MP, Geraghty P, Greene CM, O'Neill SJ, Taggart CC, McElvaney NG. Antimicrobial proteins and polypeptides in pulmonary innate defence. Respir Res 2006;7:29.
(38.) Beharka AA, Gaynor CD, Kang BK, Voelker DR, McCormack FX, Schlesinger LS. Pulmonary surfactant protein A up-regulates activity of the mannose receptor, a pattern recognition receptor expressed on human macrophages. J Immunol 2002;169:3565-73.
(39.) Eagan TM, Damas JK, Ueland T, Voll-Aanerud M, Mollnes TE, Hardie JA, et al. Neutrophil gelatinase-associated lipocalin: a biomarker in COPD. Chest 2010;138:888-95.
(40.) Chen Y, Chang L, Li W, Rong Z, Liu W, Shan R, et al. Thioredoxin protects fetal type II epithelial cells from hyperoxia-induced injury. Pediatr Pulmonol 2010;45:1192-200.
Anna Bredberg,  * Johan Gobom,  Ann-Charlotte Almstrand,  Per Larsson,  Kaj Blennow,  Anna-Carin Olin,  and Ekaterina Mirgorodskaya 
 Occupational and Environmental Medicine, University of Gothenburg, Gothenburg, Sweden;  Clinical Neurochemistry Laboratory, Department of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden.
* Address correspondence to this author at: Box 414, 405 30 Goteborg, Sweden. Fax +46-31-409728; e-mail firstname.lastname@example.org.
Received May 23, 2011; accepted October 11, 2011.
Previously published online at DOI: 10.1373/clinchem.2011.169235
 Nonstandard abbreviations: RTLF, respiratory tract lining fluid; BAL, bronchoalveolar lavage; EBC, exhaled breath condensate; PEx, particles in exhaled air; SP-A, surfactant protein A; LTQ-FT ICR, linear quadrupole ion trap-Fourier transform ion cyclotron resonance; MS , fragment ion mass spectrometry; emPAI, exponentially modified protein abundance index; CC16, Clara cell protein.
Table 1. List of proteins identified in PEx and sorted in alphabetical order by protein name. Protein name Accession Mascot no. score 14-3-3 protein [gamma] IPI00220642 46 14-3-3 protein [sigma] IPI00013890 91 14-3-3 protein [zeta]/[delta] IPI00021263 81 Actin, cytoplasmic 1 IPI00021439 465 Afamin IPI00019943 102 [[alpha].sub.1]-Acid glycoprotein 1 IPI00022429 67 [[alpha].sub.1B]-Glycoprotein IPI00022895 28 [[alpha].sub.2]-Macroglobulin IPI00478003 639 Annexin A1 IPI00218918 297 Annexin A2 IPI00455315 116 Annexin A3 IPI00024095 85 Annexin A5 IPI00329801 268 Antithrombin III IPI00032179 18 Apolipoprotein A-I IPI00021841 593 Apolipoprotein E IPI00021842 51 Arginase 1 IPI00038356 43 [[beta].sub.2]-Microglobulin IPI00004656 97 Bleomycin hydrolase IPI00219575 35 Calmodulin-like protein 3 IPI00216984 68 Cathepsin D IPI00011229 58 Ceruloplasmin IPI00017601 840 Clara cell protein (CC16) IPI00006705 261 Clusterin (apolipoprotein J) IPI00291262 65 Cofilin 1 IPI00012011 47 Complement C1q subcomponent subunit C IPI00022394 63 Complement C2 (fragment) IPI00303963 101 Complement C3 (fragment) IPI00783987 2492 Complement C4-A IPI00032258 758 Complement C4-B IPI00654875 758 Corneodesmosin IPI00386809 46 Cornifin B IPI00304903 31 Cystatin S IPI00032294 46 Deleted in malignant brain tumors 1 protein IPI00418512 196 Desmoglein 1 IPI00025753 1320 Desmoglein 4 IPI00428691 64 Epididymal secretory protein E1 IPI00940960 22 Fab 027-VL (fragment) IPI00827643 57 Fatty acid-binding protein, adipocyte IPI00215746 89 Fatty acid-binding protein, epidermal IPI00007797 118 Filaggrin 2 IPI00397801 613 FLJ00385 protein (fragment) IPI00168728 765 Galectin-3-binding protein IPI00023673 279 Glyceraldehyde-3-phosphate dehydrogenase IPI00219018 682 Glycogen phosphorylase, muscle form IPI00218130 121 Haptoglobin isoform 2 preproprotein IPI00478493 43 Heat shock protein HSP 90-[alpha] IPI00382470 72 Heat shock protein HSP 90-[beta] IPI00414676 72 Hemoglobin subunit [beta] IPI00654755 91 Hemopexin IPI00022488 282 Highly similar to complement factor B IPI00019591 88 Histidine-rich glycoprotein IPI00022371 69 Histone H2B IPI00003935 114 Histone H4 IPI00453473 76 Hornerin IPI00398625 242 Hypothetical protein LOC338667 IPI00249970 26 ICAM1 (intercellular adhesion molecule 1) IPI00008494 226 Ig [delta] chain C region IPI00163446 107 Ig heavy chain V-III region TIL IPI00382478 199 Ig [kappa] chain V-I region EU IPI00387026 546 Ig [kappa] chain V-I region Ni IPI00387106 291 Ig [kappa] chain V-I region Wes IPI00003470 173 Ig [kappa] chain V-II region TEW IPI00736885 182 Ig [kappa] chain V-III region NG9 (fragment) IPI00387116 92 Ig [kappa] chain V-IV region JI IPI00386132 109 Ig [kappa] chain V-IV region Len IPI00387120 323 Ig [lambda] protein IPI00658130 744 Ig [lambda] chain V-III region LOI IPI00385985 194 Ig [lambda] chain V-III region SH IPI00382436 100 Ig [lambda] chain V-IV region Hil IPI00382440 63 Immunoglobulin heavy chain variable region (fragment) IPI00783287 449 Immunoglobulin J chain IPI00178926 439 Inter-[alpha]-trypsin inhibitor heavy chain H1 IPI00292530 76 Isoform 1 of [[alpha].sub.1]- antichymotrypsin IPI00847635 208 Isoform 1 of [[alpha].sub.1]-antitrypsin IPI00553177 787 Isoform 1 of fibronectin IPI00022418 736 Isoform 1 of gelsolin IPI00026314 206 Isoform 1 of vitamin D-binding protein IPI00555812 253 Isoform 2 of lg [micro] chain C region IPI00896380 239 Isoform 2 of Inter-[alpha]-trypsin inhibitor heavy chain H4 IPI00218192 213 Isoform 2 of plakophilin 1 IPI00071509 190 Isoform DPI of desmoplakin IPI00013933 1742 Lactoferrin IPI00925547 289 Leukocyte antigen DR [beta] 1 chain (fragment) IPI00955443 89 Lipocalin 1 IPI00009650 143 L-lactate dehydrogenase IPI00788938 25 Lysozyme C IPI00019038 598 Macrophage mannose receptor 1 IPI00027848 212 Myosin-reactive immunoglobulin [kappa] chain variable region (fragment) IPI00384401 422 Myosin-reactive immunoglobulin light chain variable region (fragment) IPI00945366 566 Myosin-reactive immunoglobulin light chain variable region (fragment) IPI00384398 606 Neutrophil defensin 1 IPI00005721 28 Neutrophil gelatinase-associated lipocalin IPI00299547 117 Pericentrin IPI00479143 18 Phosphatidylethanolamine-binding protein 4 IPI00163563 81 Plasma protease C1 inhibitor IPI00291866 807 Polymeric immunoglobulin receptor IPI00004573 942 Profilin 1 IPI00216691 26 Prolactin-inducible protein IPI00022974 50 Proline-rich protein 4 IPI00027019 126 Protein AMBP IPI00022426 35 Protein S100-A8 (calgranulin A) IPI00007047 101 Protein-glutamine [gamma]- glutamyltransferase E IPI00300376 65 Prothrombin (fragment) IPI00019568 207 Pulmonary surfactant-associated protein A1 IPI00012889 4037 Pulmonary surfactant-associated protein B precursor IPI00296083 1615 Pulmonary surfactant-associated protein C IPI00006707 61 Putative uncharacterized 26-kDa protein IPI00942387 2448 Putative uncharacterized 26-kDa protein IPI00939805 2430 Putative uncharacterized protein DKFZp686I04196 (fragment) IPI00399007 937 Putative uncharacterized protein DKFZp686M24218 IPI00930442 533 Putative uncharacterized protein DKFZp686N02209 IPI00384938 1546 Rheumatoid factor D5 light chain (fragment) IPI00816799 205 Serotransferrin IPI00022463 4073 Serpin B12 IPI00033583 67 Serum albumin IPI00745872 16586 Similar to hCG1686089 IPI00854644 350 Small proline-rich protein 2G IPI00000849 99 Small proline-rich protein 3 IPI00082931 36 Sodium-dependent phosphate transport protein 2B IPI00007910 69 Thioredoxin IPI00216298 167 Transthyretin IPI00022432 55 Tubulin 3-2A chain IPI00013475 27 Zinc-[[alpha].sub.2]-glycoprotein IPI00166729 49 Zymogen granule protein 16 homolog B IPI00060800 29 No. of Protein name Molecular peptide size, Da matches 14-3-3 protein [gamma] 28 456 1 14-3-3 protein [sigma] 27 871 1 14-3-3 protein [zeta]/[delta] 27 899 1 Actin, cytoplasmic 1 42 052 11 Afamin 70 963 3 [[alpha].sub.1]-Acid glycoprotein 1 23 725 2 [[alpha].sub.1B]-Glycoprotein 54 809 1 [[alpha].sub.2]-Macroglobulin 164 614 14 Annexin A1 38 918 4 Annexin A2 38 808 3 Annexin A3 36 524 2 Annexin A5 35 971 7 Antithrombin III 53 025 1 Apolipoprotein A-I 30 759 9 Apolipoprotein E 36 246 1 Arginase 1 25 511 1 [[beta].sub.2]-Microglobulin 13 820 2 Bleomycin hydrolase 53 155 1 Calmodulin-like protein 3 16 937 2 Cathepsin D 45 037 1 Ceruloplasmin 122 983 19 Clara cell protein (CC16) 10 215 6 Clusterin (apolipoprotein J) 53 031 1 Cofilin 1 18 719 2 Complement C1q subcomponent subunit C 25 985 3 Complement C2 (fragment) 84 583 6 Complement C3 (fragment) 188 569 33 Complement C4-A 194 247 12 Complement C4-B 194 212 12 Corneodesmosin 52 261 1 Cornifin B 10 337 1 Cystatin S 16 489 1 Deleted in malignant brain tumors 1 protein 170 672 2 Desmoglein 1 114 702 9 Desmoglein 4 117 088 2 Epididymal secretory protein E1 16 902 1 Fab 027-VL (fragment) 12 489 1 Fatty acid-binding protein, adipocyte 14 824 1 Fatty acid-binding protein, epidermal 15 497 3 Filaggrin 2 249 296 4 FLJ00385 protein (fragment) 57 272 10 Galectin-3-binding protein 66 202 7 Glyceraldehyde-3-phosphate dehydrogenase 36 201 3 Glycogen phosphorylase, muscle form 97 487 3 Haptoglobin isoform 2 preproprotein 38 940 1 Heat shock protein HSP 90-[alpha] 98 670 2 Heat shock protein HSP 90-[beta] 83 554 2 Hemoglobin subunit [beta] 16 102 1 Hemopexin 52 385 6 Highly similar to complement factor B 143 191 6 Histidine-rich glycoprotein 60 510 1 Histone H2B 13 912 2 Histone H4 11 360 2 Hornerin 283 140 5 Hypothetical protein LOC338667 93 493 1 ICAM1 (intercellular adhesion molecule 1) 58 587 3 Ig [delta] chain C region 47 827 2 Ig heavy chain V-III region TIL 12 462 3 Ig [kappa] chain V-I region EU 11 895 5 Ig [kappa] chain V-I region Ni 12 352 5 Ig [kappa] chain V-I region Wes 11 715 2 Ig [kappa] chain V-II region TEW 12 422 2 Ig [kappa] chain V-III region NG9 (fragment) 10 836 3 Ig [kappa] chain V-IV region JI 14 737 3 Ig [kappa] chain V-IV region Len 12 746 5 Ig [lambda] protein 25 347 10 Ig [lambda] chain V-III region LOI 12 042 2 Ig [lambda] chain V-III region SH 11 500 3 Ig [lambda] chain V-IV region Hil 11 624 1 Immunoglobulin heavy chain variable region (fragment) 13 542 3 Immunoglobulin J chain 18 543 6 Inter-[alpha]-trypsin inhibitor heavy chain H1 101 782 2 Isoform 1 of [[alpha].sub.1]- antichymotrypsin 47 792 3 Isoform 1 of [[alpha].sub.1]-antitrypsin 46 878 18 Isoform 1 of fibronectin 266 034 12 Isoform 1 of gelsolin 86 043 3 Isoform 1 of vitamin D-binding protein 54 526 5 Isoform 2 of lg [micro] chain C region 52 385 6 Isoform 2 of Inter-[alpha]-trypsin inhibitor heavy chain H4 101 520 5 Isoform 2 of plakophilin 1 84 119 2 Isoform DPI of desmoplakin 334 021 23 Lactoferrin 79 812 7 Leukocyte antigen DR [beta] 1 chain (fragment) 10 988 2 Lipocalin 1 19 409 2 L-lactate dehydrogenase 25 430 1 Lysozyme C 16 982 7 Macrophage mannose receptor 1 168 870 10 Myosin-reactive immunoglobulin [kappa] chain variable region (fragment) 11 868 4 Myosin-reactive immunoglobulin light chain variable region (fragment) 11 810 6 Myosin-reactive immunoglobulin light chain variable region (fragment) 11 608 5 Neutrophil defensin 1 10 536 1 Neutrophil gelatinase-associated lipocalin 22 745 1 Pericentrin 380 644 1 Phosphatidylethanolamine-binding protein 4 26 002 1 Plasma protease C1 inhibitor 55 347 11 Polymeric immunoglobulin receptor 84 429 19 Profilin 1 15 216 1 Prolactin-inducible protein 16 847 1 Proline-rich protein 4 15 116 1 Protein AMBP 39 886 1 Protein S100-A8 (calgranulin A) 10 885 1 Protein-glutamine [gamma]- glutamyltransferase E 76 926 1 Prothrombin (fragment) 71 475 5 Pulmonary surfactant-associated protein A1 26 739 24 Pulmonary surfactant-associated protein B precursor 44 800 8 Pulmonary surfactant-associated protein C 21 381 2 Putative uncharacterized 26-kDa protein 25 886 15 Putative uncharacterized 26-kDa protein 26 214 15 Putative uncharacterized protein DKFZp686I04196 (fragment) 46 716 11 Putative uncharacterized protein DKFZp686M24218 53 071 6 Putative uncharacterized protein DKFZp686N02209 53 503 14 Rheumatoid factor D5 light chain (fragment) 12 872 3 Serotransferrin 79 280 45 Serpin B12 46 646 2 Serum albumin 71 317 79 Similar to hCG1686089 16 650 3 Small proline-rich protein 2G 8 779 2 Small proline-rich protein 3 18 598 1 Sodium-dependent phosphate transport protein 2B 77 305 2 Thioredoxin 12 015 1 Transthyretin 15 991 2 Tubulin 3-2A chain 50 274 1 Zinc-[[alpha].sub.2]-glycoprotein 34 465 1 Zymogen granule protein 16 homolog B 22 725 1 Protein name Reference 14-3-3 protein [gamma] 14-3-3 protein [sigma] 14-3-3 protein [zeta]/[delta] Actin, cytoplasmic 1 Afamin [[alpha].sub.1]-Acid glycoprotein 1 [[alpha].sub.1B]-Glycoprotein [[alpha].sub.2]-Macroglobulin Craig-Barnes et al. (21) Annexin A1 Wang et al. (22) Annexin A2 Singh et al. (23) Annexin A3 Annexin A5 Antithrombin III Apolipoprotein A-I Kim et al. (24) Apolipoprotein E Yao et al. (25) Arginase 1 [[beta].sub.2]-Microglobulin Bleomycin hydrolase Calmodulin-like protein 3 Cathepsin D Ceruloplasmin Clara cell protein (CC16) Clusterin (apolipoprotein J) Carnevali et al. (26) Cofilin 1 Vasavda et al. (27) Complement C1q subcomponent subunit C Bolger et al. (28) Complement C2 (fragment) Complement C3 (fragment) Complement C4-A Complement C4-B Corneodesmosin Cornifin B Cystatin S Deleted in malignant brain tumors 1 protein Desmoglein 1 Desmoglein 4 Epididymal secretory protein E1 Fab 027-VL (fragment) Fatty acid-binding protein, adipocyte Shum et al. (29) Fatty acid-binding protein, epidermal Filaggrin 2 FLJ00385 protein (fragment) Galectin-3-binding protein Glyceraldehyde-3-phosphate dehydrogenase Tokunaga et al. (30) Glycogen phosphorylase, muscle form Haptoglobin isoform 2 preproprotein Heat shock protein HSP 90-[alpha] Heat shock protein HSP 90-[beta] Hemoglobin subunit [beta] Hemopexin Rottoli et al. (31) Highly similar to complement factor B Histidine-rich glycoprotein Histone H2B Histone H4 Hornerin Hypothetical protein LOC338667 ICAM1 (intercellular adhesion molecule 1) Beck-Schimmer et al. (32) Ig [delta] chain C region Ig heavy chain V-III region TIL Ig [kappa] chain V-I region EU Ig [kappa] chain V-I region Ni Ig [kappa] chain V-I region Wes Ig [kappa] chain V-II region TEW Ig [kappa] chain V-III region NG9 (fragment) Ig [kappa] chain V-IV region JI Ig [kappa] chain V-IV region Len Ig [lambda] protein Ig [lambda] chain V-III region LOI Ig [lambda] chain V-III region SH Ig [lambda] chain V-IV region Hil Immunoglobulin heavy chain variable region (fragment) Immunoglobulin J chain Inter-[alpha]-trypsin inhibitor heavy chain H1 Adair et al. (33) Isoform 1 of [[alpha].sub.1]- antichymotrypsin Poller et al. (34) Isoform 1 of [[alpha].sub.1]-antitrypsin Adair et al. (33) Isoform 1 of fibronectin Isoform 1 of gelsolin Maniatis et al. (35) Isoform 1 of vitamin D-binding protein Chishimba et al. (36) Isoform 2 of lg [micro] chain C region Isoform 2 of Inter-[alpha]-trypsin inhibitor heavy chain H4 Isoform 2 of plakophilin 1 Isoform DPI of desmoplakin Lactoferrin Rogan et al. (37) Leukocyte antigen DR [beta] 1 chain (fragment) Lipocalin 1 L-lactate dehydrogenase Lysozyme C Macrophage mannose receptor 1 Beharka et al. (38) Myosin-reactive immunoglobulin [kappa] chain variable region (fragment) Myosin-reactive immunoglobulin light chain variable region (fragment) Myosin-reactive immunoglobulin light chain variable region (fragment) Neutrophil defensin 1 Neutrophil gelatinase-associated lipocalin Eagan et al. (39) Pericentrin Phosphatidylethanolamine-binding protein 4 Plasma protease C1 inhibitor Polymeric immunoglobulin receptor Profilin 1 Prolactin-inducible protein Proline-rich protein 4 Protein AMBP Protein S100-A8 (calgranulin A) Protein-glutamine [gamma]- glutamyltransferase E Prothrombin (fragment) Pulmonary surfactant-associated protein A1 Pulmonary surfactant-associated protein B precursor Pulmonary surfactant-associated protein C Putative uncharacterized 26-kDa protein Putative uncharacterized 26-kDa protein Putative uncharacterized protein DKFZp686I04196 (fragment) Putative uncharacterized protein DKFZp686M24218 Putative uncharacterized protein DKFZp686N02209 Rheumatoid factor D5 light chain (fragment) Serotransferrin Serpin B12 Serum albumin Similar to hCG1686089 Small proline-rich protein 2G Small proline-rich protein 3 Sodium-dependent phosphate transport protein 2B Thioredoxin Chen et al. (40) Transthyretin Tubulin 3-2A chain Zinc-[[alpha].sub.2]-glycoprotein Zymogen granule protein 16 homolog B Table 2. Distribution of the identified proteins according to cellular location. (a) Counts additionally annotated as: Cellular Count Count Extracellular component (total) (excl.) Extracellular 98 29 Cytoplasm 78 2 58 Membrane 60 1 45 Nucleus 36 1 26 Cytoskeleton 24 1 15 Organelle lumen 21 0 18 Cytosol 13 0 8 Mitochondrion 13 0 10 Golgi 12 0 9 Cell surface 9 0 9 Endoplasmic reticulum 8 0 7 Vacuole 8 0 6 Counts additionally annotated as: Cellular Cytoplasm Cytoskeleton Membrane component Extracellular 58 15 45 Cytoplasm 22 48 Membrane 48 14 Nucleus 32 14 22 Cytoskeleton 22 14 Organelle lumen 20 6 12 Cytosol 13 5 10 Mitochondrion 13 7 8 Golgi 12 4 12 Cell surface 9 1 9 Endoplasmic reticulum 8 1 8 Vacuole 8 1 7 (a) For each cellular component, the total number of annotated proteins (count total) and number of proteins exclusively annotated to the cellular component (count excl.) are given, as well as protein distribution among 4 major cellular compartments, i.e., number of proteins concomitantly annotated as extracellular, cytoplasm, cytoskeleton, and membrane.
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|Title Annotation:||Proteomics and Protein Markers|
|Author:||Bredberg, Anna; Gobom, Johan; Almstrand, Ann-Charlotte; Larsson, Per; Blennow, Kaj; Olin, Anna-Carin|
|Date:||Feb 1, 2012|
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